Project Summary The proto-oncogene tyrosine kinase Src is a widely expressed non-receptor tyrosine kinase that is involved in many critical cellular functions such as migration, proliferation, and survival. Although aberrant Src signaling has been implicated in cancer biology, it also contributes to non-malignant diseases such as asthma. Canonical regulation of Src involves tyrosine phosphorylation and allosteric interactions with regulatory proteins, but recent evidence has shown that oxidation of conserved cysteine residues also plays a role in Src activation and interactions with downstream targets. In the airway epithelium, innate responses to injury or airborne allergen exposure were found to involve transactivation of the epidermal growth factor receptor (EGFR) in association with Src cysteine oxidation. Our preliminary data demonstrate that Src is critical for activation of type 2 innate-immune responses to these triggers, and that oxidative activation of Src is mediated by the NADPH oxidase dual oxidase 1 (DUOX1). However, Src contains 9 cysteine residues and the specific cysteines involved in regulating Src activation, target binding and subcellular localization are unknown. Moreover, it is not known how these redox processes impact on allergen-induced type 2 responses or development of allergic asthma. We hypothesize that allergen-mediated DUOX1-dependent Src cysteine oxidation is critical for enhanced IL-33 release and activation of type 2 immune responses. To address this hypothesis, we will use in vitro and in vivo models of allergen exposure utilizing Src-deficient mice or RNAi-based methods to silence Src. We will determine the impact of redox regulation on Src on type 2 immune responses and the development of allergic asthma by assessing secretion and expression of IL-33, activation of downstream type 2 responses, and features of allergic inflammation in a house dust mite-induced model of allergic asthma. The functional consequence of cysteine oxidation for Src activation or activation of downstream signaling will be addressed using various Cys-to-Ala variants of Src transfected into lung epithelial cells. Collectively, our studies will reveal the importance of specific redox alterations in Src activation in the context of allergic asthma.